The invention relates to the processing of peptides whose structure is constrained by binding to a compound which provides a structural backbone, imparting a conformation to the peptide. In particular, the invention relate to the proteolytic treatment of such peptides, either to select protease resistant peptides or to select generated cleavage products.
The generation of molecules with high affinity and specificity for biological targets is a central problem in chemistry, biology and pharmaceutical sciences. In particular, binding ligands are important for the creation of drugs that can intervene with biological processes. The creation of ligands that bind to a chosen target ligand usually involves a process of generating a plurality of putative binding molecules and testing said molecules for their binding properties.
Polypeptides tethered to a synthetic molecular structure are known in the art (Kemp, D. S, and McNamara, P. E., J. Org. Chem., 1985; Timmerman, P. et al., Chem Bio Chem, 2005). Meloen and co-workers had used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman, P. et al., Chem Bio Chem, 2005). Methods for the generation of candidate drug compounds wherein said compounds are generated by linking cysteine containing polypeptides to a molecular scaffold as for example tris(bromomethyl)benzene are disclosed in WO 2004/077062 and WO 2006/078161.
WO2004/077062 discloses a method of selecting a candidate drug compound. In particular, this document discloses various scaffold molecules comprising first and second reactive groups, and contacting said scaffold with a further molecule to form at least two linkages between the scaffold and the further molecule in a coupling reaction.
WO2006/078161 discloses binding compounds, immunogenic compounds and peptidomimetics. This document discloses the artificial synthesis of various collections of peptides taken from existing proteins. These peptides are then combined with a constant synthetic peptide having some amino acid changes introduced in order to produce combinatorial libraries. By introducing this diversity via the chemical linkage to separate peptides featuring various amino acid changes, an increased opportunity to find the desired binding activity is provided. FIG. 7 of this document shows a schematic representation of the synthesis of various loop peptide constructs. However, the peptides produced have single specificities. Where multiple peptide loops are provided, the loops cooperate to bind to a single target.
In our copending international patent application WO2009098450 we disclose the use of biological selection technology, such as phage display, to select peptides tethered to synthetic molecular structures.
The use of proteolytic treatment to modify polypeptides, including polypeptide repertoires, is known in the art. For example, GB2428293 (Domantis Limited) describes a method for reducing the valency of peptides displayed on phage by treating the phage with protease, such that the majority of the phage display no peptides, and a proportion are monovalent.
Strategies to select stably folded proteins from repertoires of phage displayed proteins based on their resistance to proteolytic degradation have been used to improve the stability of natural proteins. Proteolytic degradation is usually restricted to unfolded proteins or highly flexible regions of folded proteins. Folded proteins are mostly resistant to proteases, because the proteolytic cleavage requires the polypeptide chain to adapt to the specific stereochemistry of the protease active site, and therefore to be flexible, accessible and capable of local unfolding.
Moreover, proteases have been used in the art for generation of desired cleavage products, for example in the production of Fc, Fab and Fv antibody fragments.
We have found that protease digestion techniques can be adapted for modifying structured polypeptides tethered to synthetic molecular scaffolds, whether displayed on phage or not. These techniques enable the production not only of protease resistant structured peptides, but also of protease cleavage products in which a plurality of polypeptides remain tethered to a scaffold.